The ability to identify and select male and female sperm has great value in the livestock industries, where there is an established market in AI of over US $2 billion per annum in the Organization for Economic Cooperation and Development (OECD) countries. This is particularly true in the dairy industry where over 80% of dairy farmers in key OECD markets impregnate their cows through artificial insemination. Sexed semen provides the opportunity to increase farmer productivity and income. For example, the availability of sexed semen has significant impact in reducing and/or eliminating the minimal returns of male dairy calves as compared to female calves.
Currently, the only commercial technique for semen sexing uses flow cytometry to sort sperm on the basis of DNA content. Bovine sperm bearing the Y chromosome have approximately 4% less DNA than sperm bearing the X chromosome. This difference, in combination with a fluorescent DNA binding dye (for example Hoechst 33342) and flow cytometry, permits purification of X chromosome bearing sperm to greater than 90% (Johnson et al., 1989). However, the ability to sort bovine sperm is currently limited to a rate of 8000 s−1 which, when each straw, or dose, contains 2×106 sperm, translates to 14 straws/hour (Sharpe and Evans, 2009). As a result, sexed semen straws generally incorporate ten-fold less sperm than unsexed straws. In addition, the sorting process itself has a negative effect on the fertility of the sperm. The reduction in the number of sperm per straw, together with reduction in sperm fertility due to the sorting process, causes a significant reduction of 14% in the conception rate for sexed sperm compared to unsexed sperm (Frijters et al., 2009). The sexed semen also has a significant price premium over unsexed sperm due to the high cost of sorting even the sub-optimal number of sperm used in the sexed semen straws. A valuable addition to the semen sexing technology would be a method to enhance the fertility of sperm so that a dose of considerably less than the approximately 2×106 sperm per straw currently used for sexed semen would achieve the same conception rate as the normal, unsexed, straws.
Such methods would also have application in swine AI where much higher doses of sperm are used in the standard AI methodology than with bovine, namely approximately 2500×106 sperm per straw. Recent work suggests that more sophisticated techniques involving deep intrauterine insemination can lower this requirement to 50-70 million (Vazquez et al., 2005; Vazquez et al., 2008). However, this reduced dose is still beyond the present commercial capability of flow cytometry sorted sperm.
The Sperm Journey in the Female Reproductive Tract
Sperm are highly specialized cells that deliver the haploid male genome to the haploid female genome contained in the oocyte. Despite this seemingly simple mission, the path to achieving this goal is highly complex. Extraordinarily large numbers of sperm are inseminated in a natural mating, for example approximately one billion sperm/oocyte in the cow. The inseminated sperm spend a variable period of time, ranging from hours to days in the different regions of the female reproductive tract (FRT). The environments that sperm encounter from ejaculation to fertilization of the oocyte also vary considerably. These environments range from the complex molecular mix added to sperm at ejaculation by the male to the various female secretions and different cell surfaces of the female epithelia (Drobnis and Overstreet, 1992).
Once sperm are deposited in the FRT, a combination of active sperm migration and female muscle contraction propels the sperm to the oocyte. During the journey through the FRT, sperm can be retained in specialized regions, most notably the cervix and oviduct (Drobnis and Overstreet, 1992). This retention presumably increases the probability that at least some sperm will be present in the oviduct at the same time as ovulation occurs. However, such retention may also act as a negative selection imposed against sperm by the female. The final phase of the sperm journey in the oviduct involves release of sperm from the isthmus region (controlled by the female) and travel to the ampulla for fertilization of the oocyte. At this time near unitary numbers of sperm are present (Drobnis and Overstreet, 1992). Fertilization itself is again a complex phenomena involving penetration of the cumulus oophorus and subsequently the zona pellucida (Katz et al., 1989). Although this complex journey is broadly similar between mammalian species, various aspects do differ.
Sperm also undergo a maturational change while resident in the FRT, known as capacitation. When sperm are ejaculated, they are not capable of fertilizing the oocyte. However, during passage through the FRT sperm gain the capacity to fertilize. Changes to sperm during passage through the FRT include alterations in membrane properties, enzyme activities and motility (Salicioni et al., 2007). Ultimately these changes enable sperm to respond to stimuli that induce the acrosome reaction and penetration of the egg. One of the important changes that occur during capacitation is alterations in the surface properties of sperm. A specialized protein-carbohydrate coating (Schroter et al., 1999) stabilizes the surface membrane, regulates capacitation (Topfer-Petersen et al., 1998), facilitates transport through the FRT (Tollner et al., 2008b), and enables attachment at the oviduct (Tollner et al., 2008a). In different species, essentially the same functions are carried out by the surface coatings, however the specific molecular components vary (Calvete and Sanz, 2007; Tollner et al., 2008a; Topfer-Petersen et al., 1998).
The Attrition of Sperm in the Female Reproductive Tract
In a natural bovine mating, approximately 1 billion sperm are inseminated yet less than 10,000 get to the oviduct and less than 10 get through to the oocyte (Mitchell et al., 1985). Why there are such large losses is poorly understood. Following coitis, greater than 80% of sperm are lost through vaginal discharge (Mitchell et al., 1985). The remaining sperm form a gradient in concentration from the cervix to the oviduct (Hawk, 1983; Hunter, 2003; Mitchell et al., 1985). In bovine, approximately 10,000 sperm arrive at the oviduct 6-8 hours after insemination (Mitchell et al., 1985). By 12 to 24 h after insemination, sperm have either been lost through back flushing, eliminated by phagocytosis or reached the oviduct (Hawk, 1983). In pigs, there is strong evidence for phagocytosis of sperm by polymorphonuclear neutrophils (PMNs), with a massive infiltration of PMNs occurring in the uterine lumen shortly after insemination (Matthijs et al., 2003). Recently, similar evidence that PMNs infiltrate the uterine lumen after insemination in cows has been published (Alghamdi et al., 2009).
How Sperm are Damaged During Passage Through the Female Reproductive Tract
Experimental evidence suggests that both motile and damaged (immotile and/or dead) sperm are lost by discharge (Lightfoot and Restall, 1971; Oren-Benaroya et al., 2007). In contrast, in vitro evidence indicates that live sperm are preferentially phagocytosed by neutrophils (Woelders and Matthijs, 2001). Several phenomena contribute to sperm damage from the FRT, although the mechanism and significance are poorly understood. Such phenomena include:
Adhesive properties of female epithelia capturing sperm and/or damaging the sperm surface, particularly mucus laden surfaces such as the cervix. This occurs by both biochemical and physical shearing (Katz et al., 1989; Mullins and Saacke, 1989).
Female secretions modulating and/or damaging the sperm surface or functionality such as flagella activity, capacitation and acrosome status. Such secretions include antibodies, complement components, molecular species affecting energy and osmotic homeostasis, signaling molecules particularly for capacitation, and also catabolic entities.
Sperm also cause damage to themselves through generation of reactive oxygen species (ROS) mainly as a by-product of mitochondrial function (de Lamirande and Gagnon, 1995; Koppers et al., 2008; Vernet et al., 2001). ROS cause loss of sperm motility and lipid peroxidation. The latter damage leads to alteration of the membrane properties such as flexibility and fluidity, and can also lead to lack of membrane integrity and/or decreased chromatin quality (Storey, 1997). Sperm are particularly sensitive to ROS-induced damage because of their membrane composition and their limited antioxidant defenses. In particular, the high proportion of polyunsaturated fatty acids (PUFA) in the surface membrane makes this membrane highly susceptible to oxidation (Jones et al., 1979). The nature of the sperm cell, with limited cytoplasmic fluid, also constrains the availability of intracellular antioxidants (Koppers et al., 2008, & ref within). In human sperm at least, there exists a strong relationship between ROS production and antioxidant protection for determining the lifespan of sperm in the absence of external damaging agents (Alvarez and Storey, 1985; Storey, 1997, 2008).
In summary, the FRT is hostile to sperm, in particular selecting for motile non-damaged sperm but also removing the vast majority of sperm. While in the FRT, sperm have to deal with a wide variety of physiological environments, mature particularly at the cell surface and respond appropriately to signals at the right time and place. Thus despite the sperm's simple mission and relatively simple construction, successful sperm have the characteristics of remaining undamaged (mainly a surface phenomena), not being phagocytosed, remaining motile (a function of mitochondria, glycolytic enzymes and flagella components), and being able to respond to signals appropriately (a surface phenomena but also involving signal transduction and effector pathways). Thus treatments to sperm that enhance the ability of sperm to remain undamaged, motile, not phagocytosed and functionally competent could reduce the number of sperm required for insemination.
Pegylation as a Method to Enhance Sperm Function
Polyethylene glycol (PEG) has the general formula H(OCH2CH2)nOH with typical molecular weights of 500-20,000 daltons. It is nonimmunogenic and soluble in aqueous solutions. The polymer is nontoxic and generally does not harm active proteins or cells.
Pegylation of proteins has been shown to improve solubility and vascular longevity, and decrease the immunogenicity of xenogeneic proteins while retaining normal protein function (Abuchowski et al., 1977a; Abuchowski et al., 1977b; Jackson et al., 1987; Senior et al., 1991; Zalipsky et al., 1994). Pegylation has also been used directly on cells to provide immune camouflage, initially for transfusion of red blood cells (Chen and Scott, 2001; Scott et al., 1997) and subsequently for other tissues such as pancreatic beta islet cells (Chen and Scott, 2001; Teramura and Iwata, 2009). For both red blood cells and pancreatic beta islet cells, the respective cell functions were preserved despite the pegylation. As the main loss of sperm within the FRT is by phagocytosis it may be possible to pegylate the sperm for protection while still preserving function.